Injectable and stable hydrogels with dynamic properties modulated by biocompatible catalysts

ABSTRACT

A hydrogel composition includes: (1) a polymer network including a first water-soluble polymer and a second water-soluble polymer that are crosslinked through dynamic bonds; and (2) a catalyst to modulate a rate of exchange of crosslinking of the polymer network.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/407,939, filed Oct. 13, 2016, the contents of which are incorporatedherein by reference in their entirety.

BACKGROUND

Hydrogels are desired for use in biomedical applications such as tissueengineering and drug delivery due to their high water content andgenerally favorable biocompatibility. Injectable hydrogels thatencapsulate bioactive therapeutics and cells have gained growinginterest because they can be administered via straightforward andminimally invasive procedures and used for cell transplantation andthree-dimensional (3D) printing of cell cultures. These hydrogels candeliver cells at a target site through a rapid sol-gel transition, andalso can provide a 3D scaffold to support cell viability post-injection.

It is against this background that a need arose to develop embodimentsof this disclosure.

SUMMARY

Hydrogels are desirable materials for biomedical applications, such ascell scaffolds for tissue regeneration and 3D printing of cell culturesas well as delivery materials for therapeutic cell transplantation anddrug delivery for disease treatment. Injectable and biocompatiblehydrogels are desired for cell transplantation to provide mechanicalprotection of cells during injection and a stable scaffold for celladhesion post-injection. Injectable hydrogels should be readily pushedthrough a syringe needle and quickly recover to a gel state, thusgenerally specifying noncovalent or dynamic crosslinking. However, adilemma can be present in the design of dynamic hydrogels: hydrogelswith fast exchange of crosslinks are more readily ejected using lessforce, but lack long-term stability; in contrast, slow exchange ofcrosslinks improves stability, but compromises injectability and thusthe ability to protect cells under flow. Some embodiments resolve thisdilemma using biocompatible catalysts to modulate the dynamic propertiesof hydrogels at different time points of application to have both highinjectability and high stability. In some embodiments, a hyaluronic acid(HA)-based hydrogel is formed through dynamic covalent hydrazonecrosslinking in the presence of a biocompatible organic catalyst. Thecatalyst accelerates the formation and exchange of hydrazone bonds,enhancing injectability, but rapidly diffuses away from the hydrogelafter injection to retard the exchange and improve the long-termstability for cell culture.

In some embodiments, a hydrogel composition includes: (1) a polymernetwork including a first water-soluble polymer and a secondwater-soluble polymer that are crosslinked through dynamic bonds; and(2) a catalyst to modulate a rate of exchange of crosslinking of thepolymer network.

In some embodiments of the hydrogel composition, the first water-solublepolymer and the second water-soluble polymer are crosslinked throughhydrazone bonds.

In some embodiments of the hydrogel composition, the first water-solublepolymer is modified with a hydrazine or hydrazide functional group, andthe second water-soluble polymer is modified with an aldehyde functionalgroup.

In some embodiments of the hydrogel composition, the first water-solublepolymer and the second water-soluble polymer are selected frompolysaccharides and proteins.

In some embodiments of the hydrogel composition, at least one of thefirst water-soluble polymer or the second water-soluble polymer ishyaluronic acid.

In some embodiments of the hydrogel composition, the first water-solublepolymer is hyaluronic acid modified with a hydrazine or hydrazidefunctional group, and the second water-soluble polymer is hyaluronicacid modified with an aldehyde functional group.

In some embodiments of the hydrogel composition, the catalyst includesan acidic functional group, a basic functional group, or both.

In some embodiments of the hydrogel composition, the catalyst is a smallmolecule organic compound.

In some embodiments of the hydrogel composition, the catalyst is aheterocyclic aromatic compound.

In some embodiments of the hydrogel composition, the catalyst is anN-heterocyclic aromatic compound substituted with an amino-containinggroup and a sulfonyl hydroxide-containing group. For example, thecatalyst can be an aminoalkyl-substituted and alkyl sulfonylhydroxide-substituted N-heterocyclic aromatic compound.

In some embodiments of the hydrogel composition, a concentration of thecatalyst in the hydrogel composition is in a range from about 0.5 mM toabout 500 mM.

In some embodiments of the hydrogel composition, a content of thepolymer network in the hydrogel composition is in a range from about 0.5wt. % to about 30 wt. %.

In additional embodiments, a method of using the hydrogel composition ofany of the foregoing embodiments includes: (1) encapsulating cells inthe polymer network of the hydrogel composition; and (2) injecting thehydrogel composition into a subject. In some embodiments of the method,injecting the hydrogel composition is performed using a syringe. In someembodiments of the method, the subject is a mammal, such as a human.

In additional embodiments, a method of forming a hydrogel compositionincludes: (1) providing a first water-soluble polymer, a secondwater-soluble polymer, and a catalyst; and (2) combining the firstwater-soluble polymer, the second water-soluble polymer, and thecatalyst in a liquid medium including water to form the hydrogelcomposition. The first water-soluble polymer is modified with ahydrazine or hydrazide functional group, and the second water-solublepolymer is modified with an aldehyde functional group. The firstwater-soluble polymer are the second water-soluble polymer arecrosslinked through hydrazone bonds to form a polymer network, and arate of exchange of crosslinking of the polymer network is modulated bythe catalyst.

In some embodiments of the method, the first water-soluble polymer andthe second water-soluble polymer are selected from polysaccharides andproteins.

In some embodiments of the method, at least one of the firstwater-soluble polymer or the second water-soluble polymer is hyaluronicacid.

In some embodiments of the method, the catalyst is a heterocyclicaromatic compound.

In some embodiments of the method, the catalyst is an N-heterocyclicaromatic compound substituted with an amino-containing group and asulfonyl hydroxide-containing group. For example, the catalyst can be anaminoalkyl-substituted and alkyl sulfonyl hydroxide-substitutedN-heterocyclic aromatic compound.

In some embodiments of the method, the catalyst is zwitterionic.

Other aspects and embodiments of this disclosure are also contemplated.The foregoing summary and the following detailed description are notmeant to restrict this disclosure to any particular embodiment but aremerely meant to describe some embodiments of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the nature and objects of some embodimentsof this disclosure, reference should be made to the following detaileddescription taken in conjunction with the accompanying drawings.

FIG. 1. Schematic representation of using diffusible organic catalyst totemporally modulate dynamic properties of hydrazone-crosslinkinghydrogels. During injection, the incorporated catalyst promotes rapidexchange of hydrazone crosslinks and rearrangement of a network tofacilitate flow. After injection, the catalyst rapidly diffuses away toslow down hydrazone exchange, resulting in the unchanged structure ofthe network with improved stability.

FIG. 2. Chemical structures of hydrazine and aldehyde modified HApolymers (left) and catalysts used to accelerate hydrazone exchange(right).

FIG. 3. Effects of catalyst 1 on the hydrazone exchange reaction andproperties of hydrazone-crosslinked HA hydrogels. a) Measuredfirst-order dependence of the rates of hydrazone formation (k₁) andcleavage (k⁻¹) on catalyst concentration. b) Oscillatory time sweep (2wt. % HA gel) showing the rate of gelation accelerated with increasingthe catalyst concentration but the equilibrium modulus of the hydrogelsremained substantially the same. c) Oscillatory frequency sweep showinghydrogel modulus independent of the incorporated catalyst concentrationbut dependent on the HA concentration. d) Stress relaxation acceleratedwith increasing the catalyst concentration but independent of the HAconcentration.

FIG. 4. a) Human Umbilical Vein Endothelial Cell (HUVEC) viability afterabout 1 h, about 4 h, and about 24 h in culture medium containing about25 and about 5 mM catalysts 1 and 2 (*p<0.05, n≥3). b) Cell viabilitywithout or with about 25 mM catalyst 2 following in vitro injection ofabout 2 wt. % hydrogel into a Petri dish through a 28-G syringe needleat about 0.05 mL min⁻¹ (*p<0.05, n≥3). c) Image of ejecting gels througha 28-G needle without clogging (Phenol red was added to color thehydrogel for visualization). d) Images of LIVE/DEAD analysis forgel-encapsulated HUVECs showing viability after injection in thepresence and absence of catalyst 2 and with no injection in the presenceof catalyst 2.

FIG. 5. a) Concentration of remaining catalyst 2 inside hydrogels overtime after immersion of catalyst-containing hydrogels in phosphatebuffered saline (PBS), showing rapid diffusion out of the hydrogels. b)Erosion kinetics of hydrogels with and without catalyst 2 at about 37°C. over about 10 days. c) Cell spreading in hydrogels at about 72 hpost-injection with initially about 25 mM of 2 during injection (Leftpanel: blue DAPI nuclear staining; middle panel: red actin cytoskeletonstaining; right panel: merged image).

FIG. 6. Size exclusion chromatography (SEC) traces of pristine HA andaldehyde-modified HA (HA-ALD). No change in molecular weightdistribution or degradation of HA was observed after modification,indicating that the mild modification procedure is compatible with HA.

FIG. 7. ¹H NMR spectrum (D₂O) of a) HA modified with hydrazine (HA-HYN)and b) HA modified with aldehyde (HA-ALD).

FIG. 8. Formation of hydrazone in model reactions at about 20 μMreactants at about 37° C. in PBS buffer (1×, pH of about 7.4) in thepresence of 0 mM, about 1 mM and about 2 mM of catalyst 1. Each datapoint represents the mean value from three independent kinetics tests.The dashed line represents the fitting of raw data using the 2^(nd)order reversible reaction kinetics model.

FIG. 9. Compression test showing remoldability of HA-hydrazone hydrogelswas improved with the addition of a catalyst. After being pressed intomolds from about 2.4 mm to about 0.6 mm in height for about 5 min, ahydrogel containing about 100 mM of 1 adopted the height of the moldwhile a hydrogel without the catalyst was not remolded (retaining itsoriginal shape) within this short time window (about 5 min).

FIG. 10. HUVEC viability at about 1 h and about 4 h after exposure toculture medium containing about 50 mM catalyst 1 and 2, and at about 1h, about 4 h, about 24 h and about 72 h after exposure to culture mediumcontaining about 5 mM catalyst 1 and 2.

FIG. 11. Nearly identical catalytic efficiency of 2 as compared to 1, asshown by nearly identically accelerated gelation time and tunablestress-relaxation rates in the presence of 1 or 2 at the sameconcentration. a) Oscillatory time sweep (2 wt. % gel) showing the rateof gelation and final storage modulus of the hydrogels in the presenceof 1 or 2. b) Oscillatory frequency sweep showing nearly identicalmodulus in the presence of 1 or 2. c) Stress relaxation showing similarrelaxation rates in the presence of 1 or 2.

FIG. 12. Hydrogel containing about 25 mM catalyst substantiallycompletely dissolved within about 12 h when the catalyst diffusion outof the gel was prevented by immersing in PBS buffer containing about 25mM catalyst.

DESCRIPTION

Shear-thinning and self-healing hydrogels encompass a class ofinjectable materials which exhibit viscous flow under an applied shearstress and time-dependent recovery upon removing the stress. Thesehydrogels can be prepared ex vivo with cells and therapeuticsencapsulated, and then flow through a needle under force and recover itsmodulus at the target site. Such injectable properties specify thehydrogels to be noncovalently or dynamically crosslinked, such aspeptide self-assembly, electrostatic attraction, hydrogen bonding,supramolecular complexation, protein interactions, and dynamic chemicalbonds. Additional, injectable hydrogels can provide cyto-protectionattributed to a shear-banding mechanism to mitigate against damage ofcell membranes. The shear-banding and plug-flow profiles localize ashear deformation within narrow regions close to a needle wall,therefore shielding most cells from extensional and shear flow.Relatively weak physical interactions and dynamic bonds with fastexchange kinetics facilitate injection of a gel, but can lead to rapiderosion of the gel. The lack of long-term stability post-injectionconstrains biological applications of such hydrogels to provide cellscaffolding or prolonged drug release. In the pursuit of an injectableand stable composition, dual-crosslinked hydrogels include an additionalcrosslinking stage post-injection. A first network is weakly crosslinkedex vivo via noncovalent interactions, and a second crosslinking isimplemented in situ using ultraviolet (UV) irradiation, temperature, orpH variation to improve mechanical properties and stability of thehydrogels. The dual-crosslinking design can increase material stability,but the secondary crosslinking stage can involve non-physiologicalconditions and can vary network structures and mechanical properties ofhydrogels, which can be incompatible with biomedical applications.

For injectable hydrogels, it is desired that the exchange dynamics ofcrosslinking of the hydrogels is modulated at different time pointsunder physiological conditions without altering an equilibrium networkstructure, chemical composition, or scaffold stiffness. That is, rapidcrosslink exchange during injection to reduce cell damage, but slowcrosslink exchange post-injection to enhance long-term networkstability. Some embodiments of this disclosure are directed to animproved approach to achieve this goal by temporally modulating theexchange dynamics of hydrazone crosslinking using an incorporatedbiocompatible, organic catalyst under physiological conditions, withoutchanging a network structure or scaffold modulus. The catalystaccelerates both the formation and exchange of hydrazone bonds tofacilitate gel shear-thinning and injectability, but quickly diffusesout of a hydrogel after injection, leading to much slower bond exchangeto stabilize a matrix. Since a catalyst accelerates the rate of bondexchange but does not affect the thermodynamic equilibrium, the networkstructure remains unchanged.

FIG. 1 illustrates a biocompatible hydrogel composition that isinjectable, and has tunable mechanical properties. Stress relaxation ofa hydrogel is modulated by including a small molecule, biocompatible,organic catalyst (or organocatalyst) in the composition. Thebiocompatible organic catalyst is used to modulate the dynamic exchangeof crosslinking of the hydrogel to tune its stress relaxation rate andrender it injectable. After injection of the hydrogel encapsulatingcells, the catalyst rapidly diffuses out of the hydrogel to slow downthe exchange of crosslinking to stabilize the hydrogel. The catalystdoes not change a structure of the hydrogel but modulates the dynamicsof its crosslinking. Thus, the catalyst allows temporal control of thehydrogel's mechanical properties to switch from fast stress relaxationfor injectability to slow stress relaxation for long-term stability,depending on the presence of catalyst. Further, a modulus and astress-relaxation rate can be independently tuned by parameters such aspolymer concentration and catalyst concentration.

Some embodiments of this disclosure are directed to compositionsincluding water-soluble polymer-based viscoelastic hydrogels thatexhibit viscous flow under shear stress and time-dependent recovery uponrelaxation under physiological conditions, such as at or about roomtemperature (about 20 to about 40° C.) and a pH of about 6 to about 8.In some embodiments, these hydrogels are formed through dynamic bonds,such as covalent hydrazone bonds resulting from hydrazine (or hydrazide)and aldehyde functional groups of water-soluble polymers.

In some embodiments, a hydrogel includes a crosslinked polymer networkthat is formed from water-soluble polymers including associativefunctional groups. In forming the network, the polymers interact withone another through their associative functional groups to form thenetwork. An associative functional group of one polymer interacts withan associative functional group of another polymer to provideintermolecular bonds or links between the polymers. In a crosslinkedpolymer network of some embodiments, polymers interact with one another(through their associative functional groups) by reversible or dynamiccovalent bonds. By crosslinking through reversible or dynamic covalentbonds, a polymer network provides a self-healing function via thesebonds, which can break preferentially (instead of other covalent bonds)during a mechanical damage event. These ‘broken’ bonds can dynamicallyassociate and dissociate to provide self-recovery.

Hydrazone formation is an efficient, biocompatible chemistry that can beused for bioconjugation under physiological conditions. The reactionrate of formation and dissociation of a hydrazone bond can be dependenton structures of aldehyde and hydrazine (or hydrazide) used to form thehydrazone bond. Such dynamic nature makes hydrazone an appealingcrosslinking chemistry to form viscoelastic hydrogels with tunablemechanical properties by structure variation. A variety of compounds canbe used as catalysts to accelerate hydrazone formation, includingacid/base catalysts in accelerating a rate-limiting dehydration stage.These catalysts can promote the rate of hydrazone exchange, thusenhancing the ability of hydrazone-crosslinking hydrogels to flowthrough a syringe

One aspect of some embodiments of this disclosure is to providehydrazine-modified, hydrazide-modified, and aldehyde-modifiedwater-soluble polymer derivatives with biomedicine and other usages andwhich contain adjustable molecular structures (e.g., of side chains orbackbone) to yield tunable mechanical properties. Another aspect of someembodiments of this disclosure is to provide hydrazone bond crosslinkedhydrogels composed of hydrazine-modified (or hydrazide-modified) andaldehyde-modified polymer derivatives.

In some embodiments, water-soluble polymers, such as including sidecarboxyl groups, are used as starting materials, and hydrazine-modified,hydrazide-modified, and aldehyde-modified polymers are synthesizedthrough chemical modification.

Modified polymers of some embodiments of this disclosure are representedby the following chemical structures (I), (II), and (III):P—NH—NH₂  (I)P′—(C═O)—NH—NH₂  (II)P″—(C═O)—H  (III)

Chemical structure (I) represents a water-soluble polymer P that ismodified with a moiety containing a nitrogen-nitrogen covalent bond and,more specifically, with a hydrazine functional group (—NH—NH₂). Ingeneral, the hydrazine functional group can be included in a side chainor as part of a backbone of the polymer P. Also, the polymer P can bemodified with a single hydrazine functional group, or can be modifiedwith multiple hydrazine functional groups.

Chemical structure (II) represents a water-soluble polymer P′ that ismodified with a moiety containing a nitrogen-nitrogen covalent bond and,more specifically, with a hydrazide functional group (—(C═O)—NH—NH₂). Ingeneral, the hydrazide functional group can be included in a side chainor as part of a backbone of the polymer P′. Also, the polymer P′ can bemodified with a single hydrazide functional group, or can be modifiedwith multiple hydrazide functional groups.

Chemical structure (III) represents a water-soluble polymer P″ that ismodified with a carbonyl (C═O)-containing moiety and, more specifically,with an aldehyde functional group (—(C═O)—H). In general, the aldehydefunctional group can be included in a side chain or as part of abackbone of the polymer P″. Also, the polymer P″ can be modified with asingle aldehyde functional group, or can be modified with multiplealdehyde functional groups.

The polymers P, P′, and P″ in chemical structures (I), (II), and (III)can be the same or different, and can be selected from a range ofwater-soluble (or hydrophilic) polymers. Examples of suitable polymersinclude polysaccharides, such as chondroitin sulfate, dermatan, heparin,heparan, alginic acid, hyaluronic acid, dermatan sulfate, pectin,carboxymethyl cellulose, carboxymethyl chitosan, and their salts.Additional examples of suitable polymers include proteins, such ascollagen protein, gelatin, elastin, decorin, laminin, fibronectin, andtheir salts. Further examples of suitable polymers include syntheticpolymers, such as polyacrylic acid, polymethylacrylic acid, polyasparticacid, polytartaric acid, polyglutamic acid, polyfumaric acid,poly(N-isopropylacrylamide), polyacrylamide, poly(2-oxazoline),polyethylenimine, polymethacrylate, and their salts.

For example, hyaluronic acid (HA) is an anionic glycosaminoglycan widelydistributed in many tissues including cartilage and brain. Due to itsbiocompatibility and diverse biological functions, HA is a desirablepolymer for the formation of hydrogels with desired morphology,mechanical properties, and bioactivity for biomedical applications.

In some embodiments, the polymers P, P′, and P″ can be modified byincluding hydrazine, hydrazide, and aldehyde functional groups in sidechains of the polymers P, P′, and P″, in which case the modifiedpolymers are represented by the following chemical structures (Ia),(IIa), and (IIIa):P-L-NH—NH₂  (Ia)P′-L′-(C═O)—NH—NH₂  (IIa)P″-L″-(C═O)—H  (IIIa)

Linkers L, L′, and L″ in chemical structures (Ia), (IIa), and (IIIa) canbe the same or different. In some embodiments, L, L′, and L″ can be, orcan include, a C₁-C₂₀, C₁-C₁₅, C₁-C₁₀, C₁-C₅ C₂-C₁₅, C₂-C₁₀, or C₂-C₅alkylene or heteroalkylene group, optionally substituted with 1-5 C₁-C₆alkyl groups or other substituent groups. For example, L, L′, and L″ canbe, or can include, —(CH₂)_(n)—, where n is from 1-20, 1-15, 1-10, 1-5,2-15, 2-10, or 2-5. L, L′, and L″ also can be, or can include, one ormore aromatic rings, such as benzene rings, and also can be, or caninclude, one or more heterocylic rings, such as triazole rings. L, L′,and L″ also can be, or can include, one or more amide linkages(—(C═O)—NR—), where R is hydrogen, a C₁-C₆ alkyl group, or anothersubstituent group. In some embodiments, L, L′, and L″ can be, or caninclude, a polyether group, such as —[(CHR)_(x)O]_(y), where x is from1-10, 1-5, 2-10, or 2-5, y is from 1-20, 1-15, 1-10, 1-5, 2-15, 2-10, or2-5, and R is hydrogen, a C₁-C₆ alkyl group, or another substituentgroup.

For example, if the polymers P, P′, and P″ are hyaluronic acid, specificexamples of the modified polymers of some embodiments include thefollowing:

In chemical structures (IV) through (VI), i is an integer greater thanor equal to 1, j is an integer greater than or equal to 0, and a sum ofi and j is an integer greater than or equal to 1, such as i+j≥2, ≥5,≥10, ≥15, ≥20, ≥50, ≥100, or ≥200.

Hydrogel compositions of some embodiments of this disclosure can beformed by mixing or otherwise combining aqueous solutions of at leasttwo different modified water-soluble polymers, a first one of which is ahydrazine or hydrazide-modified polymer, such as given by chemicalstructure (I) or (II) (or (Ia) or (IIa)), and a second one of which isan aldehyde-modified polymer, such as given by chemical structure (III)(or (IIIa)). Mixing induces crosslinking of the polymers through theformation of hydrazone bonds, yielding a crosslinked polymer networkdispersed in water, and where the hydrazone bonds can be represented as:

Mixing of the polymers can be performed in conjunction or sequentiallywith at least one biocompatible, small molecule catalyst, such as onehaving a molecular weight of no greater than about 1 kDa, no greaterthan about 900 Da, or no greater than about 500 Da. Examples ofcatalysts include those including acidic functional groups, basicfunctional groups, or both. For example, suitable catalysts can includeheterocyclic aromatic compounds, such as aminoalkyl-substitutedN-heterocyclic aromatic compounds like 2-(aminomethyl)benzimidazole andits sulfonated or other derivatives. As another example, suitablecatalysts can include aromatic acid compounds such as anthranilic acidand its derivatives like 2-aminobenzenephosphonic acid. As anotherexample, suitable catalysts can include aromatic alcohol compounds, suchas 2-aminophenols and its derivatives. Suitable catalysts can becationic, anionic, or zwitterionic under physiological conditions (e.g.,pH of about 7.4). Suitable catalysts can have a pK_(a) (logarithm of itsacid dissociation constant) in a range of about 6 to about 9, about 6 toabout 8.5, about 6 to about 8, about 6.5 to about 8, or about 7 to about8. Mixing of the polymers can be performed in conjunction orsequentially with cells or bioactive therapeutics.

In some embodiments, a polymer content of a hydrogel composition can bein a range from about 0.1 wt. % to about 30 wt. %, about 0.5 wt. % toabout 30 wt. %, about 0.5 wt. % to about 25 wt. %, about 0.5 wt. % toabout 20 wt. %, about 0.5 wt. % to about 15 wt. %, or about 1 wt. % toabout 10 wt. %, with a remaining content including water as a liquidmedium, one or more catalysts, and cells or bioactive therapeutics.

In some embodiments, a catalyst concentration or loading of a hydrogelcomposition can be in a range from about 0.1 millimolar (mM) to about 1molar (M), about 0.5 mM to about 500 mM, about 0.5 mM to about 400 mM,about 0.5 mM to about 300 mM, about 0.5 mM to about 200 mM, about 0.5 mMto about 100 mM, about 1 mM to about 100 mM, about 1 mM to about 5 mM,or about 5 mM to about 100 mM.

EXAMPLE

The following example describes specific aspects of some embodiments ofthis disclosure to illustrate and provide a description for those ofordinary skill in the art. The example should not be construed aslimiting this disclosure, as the example merely provides specificmethodology useful in understanding and practicing some embodiments ofthis disclosure.

Temporally Modulating Exchange Kinetics of Dynamic Hyaluronan Hydrogelsby a Biocompatible Organocatalyst to Achieve Both High Injectability andHigh Stability

Hyaluronic acid (HA) is chosen as a polymer backbone to form hydrogels,since it is a biocompatible, naturally abundant polysaccharide in humantissue and plays an important role in many biological processes. HA is arelatively sensitive natural polymer that can degrade under acidic,basic, and oxidative conditions. Therefore, a mild strategy is developedto modify HA with hydrazine and aldehyde groups without backbonedegradation (FIG. 6): a pre-determined amount of alkyne functionalitieswere attached to HA via carbodiimide coupling, which were thenfunctionalized with hydrazine and aldehyde groups via copper catalyzed“click” reaction (FIG. 2).

HA-hydrazone hydrogels can be formed by mixing dilute solutions ofhydrazine-modified HA and aldehyde-modified HA in phosphate bufferedsaline (PBS, pH of about 7.4) at about 37° C. HA with about 60 kDamolecular weight is selected, and about 127% of its carboxylate groupsare modified with aldehyde or hydrazine (FIG. 2) to evaluate the effectof incorporated catalyst on the properties of formed hydrogels and theirefficacy for cyto-protection during injection. These HA hydrogelsexhibited stress-relaxation behavior as a result of the dynamic exchangeof the hydrazone crosslinking. And their mechanical properties can betuned using several parameters, such as HA concentration, chemicalstructure of hydrazone, molecular weight (MW) of HA, and degree ofmodification on HA.

2-(aminomethyl)benzimidazole (1 in FIG. 2) is an efficient catalyst toaccelerate hydrazone formation owing to facilitated intramolecularproton transfer in the transition state. The rates for hydrazoneformation (k₁) and cleavage (k⁻¹) in the presence of 1 were measuredusing model reactions between hydrazine and aldehyde and modeled for areversible second-order reaction (FIG. 8), and the equilibrium constant(K_(eq)) was calculated from these rate constants. 1 exhibited highcatalytic activity and first-order dependence on the reaction rate (FIG.3a ). Both k₁ and k⁻¹ showed about 24, about 47, and about 94-foldenhancement in the presence of about 25, about 50, and about 100 mM of1, respectively (Table 1), and K_(eg) remained substantially constantand independent of the catalyst concentration. The gelation time of theHA-hydrazone hydrogels was dramatically shortened in the presence of 1.For an about 2 wt. % HA hydrogel, equilibrium gelation was reached inabout 30 min without 1 (FIG. 3b ). In the presence of about 25 mM of 1,gelation occurred in less than about 60 s and reached a final plateaushear modulus of about 800 Pa in about 5 min (FIG. 3b ). Increasing thecatalyst loading to about 100 mM further shortened the time to reachequilibrium to about 3 min (FIG. 3b ). As shown in the frequency sweepexperiment (FIG. 3c ), stable gels were formed with a plateau storagemodulus (G′) of about 1 kPa, which is more than an order of magnitudehigher than the loss modulus (G″). The hydrogel plateau modulus remainedsubstantially the same regardless of the catalyst loading (examplesshown using about 2 wt. % HA gels at 0, about 25, about 50, and about100 mM catalyst loadings in FIG. 3c ), confirming that the catalyst didnot affect the equilibrium network structure or hydrogel modulus. Incontrast, altering the HA polymer composition from about 1 to about 4wt. % increased the plateau modulus (FIG. 3c ). The dynamic exchange ofhydrazone crosslinks allowed the HA hydrogels to exhibitstress-relaxation behavior (FIG. 3d ), which correlates to the ease offlowing under applied force. The rate of stress relaxation underconstant strain was quantified by the time for the initially measuredstress to relax to half of its original value, τ_(1/2). The catalystremarkably enhanced the rate of stress-relaxation for these hydrogels,decreasing τ_(1/2) from about 75 min without 1 to about 10 min and about3 min with about 25 mM and about 100 mM of 1, respectively (FIG. 3d ).Accelerated hydrazone exchange by an incorporated catalyst also allowedthese hydrogels to be readily remolded macroscopically (FIG. 9),facilitating their injection and re-processing. Furthermore, alteringthe hydrogel stiffness by tuning the HA concentration did not impact thestress-relaxation rate, as shown by the same stress-relaxation profilesbetween about 1, about 2, and about 4 wt. % HA hydrogels in FIG. 3d .Therefore, the design provides a strategy to tune the modulus and rateof stress relaxation, two important mechanical properties for dynamichydrogel materials, independently and continuously by polymerconcentration and catalyst concentration, respectively.

High cyto-compatibility is desired for catalysts used in this design forbiomedical applications. In view of the cationic nature of catalyst 1, asulfonated derivative of 1 is synthesized, thus converting it to azwitterionic form, 2, to impart enhanced biocompatibility.Cyto-compatibility of both 1 and 2 were evaluated using two-dimensional(2D) cultures of Human Umbilical Vein Endothelial Cells (HUVECs). HUVECswere chosen as an example test cell line because they are a clinicallyrelevant human cell type that has been broadly explored for tissueengineering and regenerative medicine applications. Significant celldeath was observed in the presence of about 25 mM of 1 within severalhours (FIG. 4a ). In sharp contrast, 2 exhibited highcyto-compatibility, with about 85% cells remaining viable after about 24h exposure at about 25 mM and negligible cell death at about 5 mM evenafter about 3 days (FIG. 4a and FIG. 10). Such dramatically improvedcyto-compatibility of 2 was attributed to its zwitterionic nature. Ofnote, 2 was found to exhibit similar catalytic efficiency to 1, andyielded almost identically accelerated gelation time and tunablestress-relaxation rates as 1 at the same catalyst concentration (FIG.11).

Evaluation was performed on the effect of catalyst 2 incorporated inHA-hydrazone hydrogels on the protection of encapsulated cells duringinjection. HUVECs were homogeneously encapsulated in about 2 wt. %hydrogels containing about 25 mM of 2 by rapidly mixing the cellssuspended in HA solutions with catalyst and transferring to a syringe.Hydrogel was rapidly formed in the syringe and ejected through a28-gauge syringe needle. A syringe pump was used for all cell injectionexperiments to ensure consistent flow rate for accurate comparisonbetween all samples. In the presence of about 25 mM of 2, the hydrogelscan be readily ejected through the thin needle without clogging (FIG. 4c). In contrast, in the absence of a catalyst, the same hydrogelexperienced high resistance to flow.

After injection, cells encapsulated in the hydrogels were incubated forabout 20 min, and cell viability was then analyzed using a LIVE/DEADstaining assay. In the control gel without catalyst 2, the injection ledto significant cell death with about 65% viability, presumably due tomembrane damage during injection (FIG. 4b,d ). In contrast, the presenceof about 25 mM of 2 significantly increased the cell viability to about87% after injection, which is similar to the viability of cellsencapsulated in identically prepared hydrogels without injection (FIG.4b,d ). This result strongly indicated that the enhanced networkdynamics of hydrogels in the presence of the catalyst improvedinjectability and provided better protection for encapsulated cellsduring flow.

Long-term stability of hydrogel scaffolds is often desired to providesupport for cell adhesion and growth after cell injection. Rapidhydrogel erosion is a challenge for many dynamic and injectablehydrogels. It is proposed that rapid passive diffusion of the dissolvedsmall molecule catalyst away from the hydrogel post-injection would slowdown the dynamic exchange of hydrazone crosslinks and thus enhance thehydrogel stability. Therefore evaluation was performed to monitor thediffusion of 2, which was incorporated in the hydrogel at either about25 mM or about 50 mM initial concentration, into the buffer solutionafter injection by monitoring its absorption at about 281 nm. Afterabout 50 μL hydrogel was injected and immersed in about 1 mL PBS, about60% of 2 diffused out of the hydrogel within about 1 h, and less thanabout 1% remained inside the gel after about 8 h (FIG. 5a ). To examinewhether the rapid diffusion and thus removal of catalyst can increasethe long-term stability of the hydrogels, comparison is performed of theerosion rate of hydrogels with different initial concentrations ofincorporated catalyst. Hydrogels without incorporated catalyst exhibitedan initial erosion rate of about 3.5% per day (over about 10 days), asdetermined by fitting the erosion data to a zero-order kinetic model,and about 65% of the hydrogel mass was retained after about 10 days.Hydrogels initially containing about 25 mM or about 50 mM of catalyst 2had erosion rates of about 3.8 and about 4.1% per day respectively,similar to those without a catalyst (FIG. 5b ), presumably due to rapiddiffusion of 2 out of the hydrogels. In contrast, when diffusion of 2away from the hydrogel was prevented by immersing the hydrogel into abuffer solution containing about 25 mM of 2, the hydrogel was completelydissolved within about 12 h (FIG. 12). Therefore, the strategytemporally modulates the exchange rate of dynamic crosslinking,achieving the desired short-term injectability and long-term stabilityat different stages of the application from the same hydrogel network.

To test the ability of the HA hydrogels to support cellular growth as ascaffold after injection, cell-adhesive Arg-Gly-Asp (RGD) peptide motifsare attached to the hydrazine-functionalized HA polymer. HUVECs wereencapsulated within the RGD-presenting HA-hydrazone hydrogels in thepresence of about 25 mM of 2, injected through a 28-gauge needle into aPetri dish, and cultured under physiological conditions in EBM-2(Endothelial Growth Basal Medium) for about 3 days. Cell morphology wasanalyzed by staining and imaging of the cell nuclei and actincytoskeleton. HUVECs encapsulated within the hydrogels demonstrated aspread morphology (FIG. 5c ).

In summary, this example sets forth a strategy to temporally modulatethe exchange kinetics of dynamically crosslinked hydrogels using abiocompatible organic catalyst to provide high injectability and highstability at different stages of cell delivery. In this strategy, thecyto-compatible sulfonated amino-benzimidazole functions as an effectivecatalyst to temporally accelerate the rates of formation and exchange ofdynamic covalent hydrazone crosslinks in HA-based hydrogels. As aresult, the presence of the catalyst enhances the rates of gelation andstress-relaxation, but does not alter the hydrogel network structure norits storage modulus, which allowed independent and continuous tuning ofthe stiffness and stress-relaxation rate of the hydrogels by varying thepolymer concentration and catalyst loading, respectively. Theaccelerated exchange of crosslinking led to enhanced injectability ofthe hydrogels and improved cell protection during injection. As thecatalyst rapidly diffused out of the hydrogels after injection, thehydrogels gained high stability and slow erosion post-injection toprovide a long-term, cell-adhesive scaffold for cell culture. Thiseffective design bestows hydrogels with both high injectability andstability, two often conflicting but desired properties of dynamichydrogels used for biomedical applications. Considering the broadapplications of biocompatible hydrazone chemistry, this strategy can beapplicable to a wide range of dynamic hydrogel materials for therapeuticcell delivery and 3D printing of encapsulated cell scaffolds.

Experimental Section

Materials. Sodium hyaluronate (about 60 kDa) was purchased fromLifecore. All other chemicals were obtained from commercial sources andused as received unless otherwise noted.2-(2-(2-(azidoethoxy)ethoxy)ethoxy)acetaldehyde (S1) was preparedaccording to reported procedures. Analytical thin-layer chromatography(TLC) was carried out using about 0.2 mm silica gel plates (silica gel60, F254, EMD chemical).

Characterizations. ¹H and ¹³C NMR spectra were recorded using 400 VarianNMR spectrometers. Chemical shifts are reported in ppm using theresidual protiated solvent as an internal standard (CDCl₃ ¹H: about 7.26ppm and ¹³C: about 77.0 ppm; D₂O ¹H: about 4.79 ppm; DMSO-d6 ¹H: about2.50 ppm and ¹³C: about 39.5 ppm). High-performance liquidchromatography-mass spectrometry (HPLC-MS) was performed inacetonitrile/water containing about 0.1% formic acid on an Alliancee2695 Separations Module using an)(Bridge 10 μm C18 column in serieswith a 2489 UV/Visible Detector and an Acquity QDa Detector (all fromWaters Corporation). Rheological characterization was performed using anAR-G2 controlled stress rheometer at about 37° C. All measurements wereperformed using an about 20 mm cone plate geometry and analyzed usingTRIOS Software. UV-Vis spectra were recorded on a SpectraMax M2Spectrophotometer. Aqueous size exclusion chromatography (SEC) wascarried out using Shimadzu LC-20AD high performance liquidchromatography with a refractive index detector.

1. Synthetic Procedures

N-(3-azidopropyl)-2-hydrazineylacetamide (S2)

N-(3-azidopropyl)-2-hydrazineylacetamide was synthesized according toreported procedures with modification. Tri-Boc-hydrazinoacetic acid(about 2.5 g, about 6.4 mmol, about 1 eq.), azidopropyl amine (about0.84 g, about 8.3 mmol, about 1.3 eq.),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (about 1.6 g, about 8.3mmol, about 1.3 eq.), and 4-dimethylaminopyridine (about 0.16 g, about1.3 mmol, about 0.2 eq.) were dissolved in about 20 mL methylenechloride (DCM). The solution was stirred at room temperature overnight.The solvent was removed under reduced pressure, and the residue waspurified by silica gel chromatography (ethyl acetate/hexane, about 1:1v/v) to obtain the Boc-protected product as a colorless oil (about 2.8g, about 92% yield). The isolated Boc-protected product was thendissolved in about 10 mL of about 4 M HCl in dioxane. After stirring atroom temperature for about 4 h, the product precipitated from thesolution as a sticky yellow solid (about 1.2 g, about 95% yield). ¹H NMR(400 MHz, D₂O) δ 3.71 (s, 2H), 3.33 (t, J=6.7 Hz, 2H), 3.26 (t, J=6.7Hz, 2H) 1.74 (dd, J=6.7 Hz, 2H). ¹³C NMR (100 MHz, D₂O) δ 169.57, 50.65,48.72, 36.70, 27.64. MS (ESI) m/z [M⁻H⁺]: 173.05.

Catalyst 2

2-(Aminomethyl)benzimidazole dihydrochloride (about 600 mg, about 2.7mmol, about 1 eq.) and trimethylamine (about 2.3 mL, about 16.4 mmol,about 6 eq.) were dissolved in about 12 mL tetrahydrofuran (THF).Di-tert-butyl dicarbonate (about 595 mg, about 2.7 mmol, about 1 eq.),dissolved in about 3 mL THF, was then added dropwise to the mixture at0° C. The solution was allowed to warm up to room temperature andstirred at room temperature overnight. The mixture was then filtered andthe filtrated was concentrated. The crude product was purified by silicagel chromatography (DCM/methanol, about 93:7 v/v) to obtain theBoc-protected product as white solid (about 550 mg, about 82% yield).This Boc-protected benzimidazole (about 200 mg, about 0.81 mmol, about 1eq.), 1,3-propane sultone (about 109 mg, about 0.89 mmol, about 1.1eq.), and potassium hydroxide (about 50 mg, about 0.89 mmol, about 1.1eq.) were dissolved in about 3 mL dimethylformamide (DMF). The solutionwas stirred at about 60° C. for about 2d. The solvent was removed underreduced pressure and the residue was purified by silica gelchromatography (DCM/methanol, about 4:1 v/v) to obtain the product aswhite solid (about 160 mg, about 54% yield). ¹H NMR (400 MHz, DMSO-d6) δ7.67 (d, J=7.9 Hz, 1H), 7.59 (d, J=7.7 Hz, 1H), 7.52 (t, J=5.9 Hz, 1H),7.25 (m, 2H), 4.47 (d, J=5.9 Hz, 2H), 4.40 (t, J=7.6 Hz, 2H), 2.46 (t,J=6.9 Hz, 2H), 2.01 (m, 2H), 1.38 (s, 9H). ¹³C NMR (100 MHz, DMSO-d6) δ163.74, 156.28, 152.53, 135.41, 123.38, 122.96, 118.56, 111.55, 79.14,48.72, 43.00, 37.58, 28.86, 26.43. MS (ESI) m/z [M⁻H⁺]: 370.2.

The isolated Boc-protected product was then dissolved in about 3 mL ofabout 4 M HCl in dioxane. After stirring at room temperature overnight,the product precipitated from the solution. The solid was then washedwith ether 3 times and isopropyl alcohol once to obtain catalyst 2 asyellowish solid (about 140 mg, about 94% yield). ¹H NMR (400 MHz,DMSO-d6) δ 7.89 (d, J=7.8 Hz, 1H), 7.78 (d, J=8.5 Hz, 1H), 7.52 (m, 2H),4.58 (m, 4H), 2.54 (t, J=6.7 Hz, 2H), 2.11 (m, 2H). ¹³C NMR (100 MHz,DMSO-d6) δ 147.83, 136.12, 134.02, 125.39, 125.30, 117.23, 112.92,48.05, 43.71, 34.44, 26.02. MS (ESI) m/z [M⁻H⁺]: 270.1

Hyaluronic Acid (HA) Modification

HA Modification with Alkyne

Sodium hyaluronate was dissolved in 2-(N-Morpholino)ethanesulfonic acid(MES) buffer (about 0.2 M, pH of about 4.5) to a concentration of about10 mg/mL. To this solution, N-hydroxysuccinimide (about 223 mg per gramof HA, about 0.8 eq. to the HA dimer unit),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (about 372 mg per gramHA, about 0.8 eq.) and propargyl amine (about 128 μL per gram HA, about0.8 eq.) were added successively. After adjusting pH to about 6, themixture was stirred at room temperature for about 4 h. The solution wasthen dialyzed against de-ionized (DI) water for about 3 d andlyophilized to give a white powder. The degree of modification wasquantified by ¹H NMR spectroscopy after “click” functionalization.

HA Modification with Hydrazine and Aldehyde

HA-alkyne (about 300 mg) was dissolved in PBS (pH of about 7.4) at 2 wt.% followed by the addition of azido-aldehyde (S1) or azido-hydrazine(S2) (about 100 mg, about 1 eq. to HA dimer unit). The solution was thenbubbled with N₂ for about 30 min. Copper (II) sulfate pentahydrate(about 0.76 mg, about 0.004 eq. to HA dimer unit) and sodium ascorbate(about 8.7 mg, about 0.06 eq. to HA dimer unit) were dissolved in DIwater, bubbled with N₂, and added to HA solution. After stirring at roomtemperature for about 1 d, the mixture was dialyzed against DI water forabout 3 d and lyophilized. The degree of modification on HA wasquantified using ¹H NMR spectroscopy by integration of the proton signalon triazole group (δ=7.85, 1H) relative to that of the methyl groups onN-acetylglucosamine of HA backbone (δ=1.8, 3H). ¹H NMR integrationindicated that about 12% of the carboxylate groups on the HA backbonehave been functionalized (FIGS. 6-7).

HA Modification with RGD

RGD-HA was prepared by coupling the oligopeptide GGGGRGDSP (PeptidesInternational) to HA using carbodiimide chemistry. Sodium hyaluronate(about 250 mg) was dissolved in MES buffer (about 0.1 M, pH of about6.5) to a concentration of about 10 mg/mL. To this solution,N-Hydroxysulfosuccinimide sodium salt (about 51 mg),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (about 91 mg) and RGDpeptide (about 60 mg) were added successively. The mixture was stirredat room temperature for about 20 h and quenched by the addition ofhydroxylamine hydrochloride (about 180 mg). The solution was thendialyzed against DI water for about 3 d and lyophilized to give a whitepowder. The coupling efficiency using this procedure was characterizedby bicinchoninic acid (BCA) assay, which indicated that about 6% of thecarboxylate groups on the HA backbone have been functionalized. Thiscorresponds to the density of about 1.5 mM RGD in an about 2 wt. % HAgel. RGD-HA was then modified with hydrazine groups using the sameprocedure as described in the previous section.

2. Hydrogel Preparation

HA-hydrazine and HA-aldehyde were first solubilized in PBS (1×, pH ofabout 7.4) at 5 wt. % respectively. Catalyst 1 and 2 were dissolved inPBS (1×, pH of about 7.4) and adjusted pH to about 7.4 at a stockconcentration of about 250 mM. HA hydrogels were prepared by mixingHA-hydrazine, catalyst, and HA-aldehyde stock solutions successively athydrazine to aldehyde (molar) ratio of about 1:1. The volumes of stocksolutions were adjusted to make gels with different formulations.

3. Rheological Tests

Rheological characterization was performed on a stress-controlledrheometer (AR-G2, TA instrument) using a 20 mm cone plate. About 45 μLof a sample was loaded immediately onto the rheometer after mixing togel in situ on the rheometer and a humidity chamber was secured in placeto prevent dehydration. All the tests were performed at about 37° C.Time sweeps were performed at about 1 Hz at about 1% constant strain.Frequency sweeps were performed from about 0.1 to about 10 Hz at about1% constant strain. Stress relaxation experiments were performed atabout 10% strain.

4. Kinetic Measurements on Hydrazone Exchange

The equilibrium constant (K_(eq)) and rate constants for hydrazoneformation (k₁) and cleavage (k⁻¹) between aldehyde S1 and hydrazine S3as model compounds with different concentrations of catalyst 1 weremeasured by HPLC. S3 was synthesized by couplingN-(2-aminoethyl)-4-amino-1,8-naphthalimide with hydrazine usingcarbodiimide chemistry. About 2 mM stock solutions of hydrazine andaldehyde were prepared in a PBS buffer (1×, pH of about 7.4). An about250 mM stock solution of catalyst 1 was prepared in PBS as described inthe previous sections.

For the reactions performed in the absence of catalyst, about 10 μL ofhydrazine and about 10 μL of aldehyde stock solutions were added toabout 980 μL PBS buffer (1×, pH of about 7.4). For the reactionsperformed in the presence of catalyst 1, the appropriate amount ofcatalyst 1, hydrazine, and aldehyde were sequentially added to PBSbuffer (1×, pH of about 7.4) from their stock solutions, maintaining thesame concentrations for hydrazine and aldehyde as those without acatalyst. The reactions were performed at about 37° C. in a water bathand followed using HPLC with detection at about 420 nm absorption (HPLCconditions: XBridge 10 μm C18 column; about 40% acetonitrile in PBSbuffer (pH of about 7) isocratic elution in about 7 min at a flow rateof about 0.34 mL/min. The concentrations and conversions were calculatedfrom the integrals of the HPLC signals. The kinetic data of hydrazoneformation were fitted to a kinetic model for 2^(nd) order reactionsusing Matlab (FIG. 8).

5. Catalyst Diffusion and Hydrogel Erosion Study

HA hydrogels containing 0, about 25, and about 50 mM of catalyst 2 wereprepared as described in the previous section. About 50 μL of thehydrogel was prepared in the syringe and ejected through a 28-gaugesyringe needle into separate centrifuge tubes for the erosion test. Thehydrogel was allowed to cure for about 30 min before immersed in buffersolution. These gels were then immersed in about 1 mL PBS and incubatedat about 37° C. All the buffer solutions were collected and replaced byfresh PBS at about 1 h, about 4 h, about 8 h, about 1 d, about 2 d,about 3 d, about 5 d, about 7 d and about 10 d. The remaining hydrogelswere degraded by about 2 mg mL⁻¹ hyaluronidase to allow determination ofremaining hydrogel content for data normalization. Four replicates wereprepared in each group. Catalyst diffusion was quantified using UVmeasurement by monitoring absorption at about 281 nm. HA erosion wasquantified by uronic acid assay according to reported procedures.

6. Cell Culture Experiments

Cell Culture

Human Umbilical Vein Endothelial Cells (HUVECs) were obtained fromLonza. HUVECs were expanded in growth medium composed of about 10% fetalbovine serum, about 1% penicillin/streptomycin in EBM-2. The medium waschanged every about 3 d and the cells were passaged at about 70%confluency. Cells were used at passage 5 or lower for all experiments.

Catalyst Toxicity

HUVECs pre-seeded in 96-well plates at about 10⁴ cells/well wereincubated with catalysts 1 and 2 for about 1 h, about 4 h, about 24 hand about 72 h. Afterwards, the cells were washed with PBS, the cellviability MTT assay was carried out to determine the cell viabilitiesrelative to the control cells incubated with the same volume of PBS.

In Vitro Cell Injection and Quantification of Viability

Cell suspension was first mixed with HA-hydrazine stock solutions beforefurther mixing with catalyst and HA-aldehyde stock solutions. All invitro injection experiments were performed with about 30 μL gel volumecontaining about 3×10⁴ cells. For cell injection, the final mixing stagewas performed in the barrel of an about 1 mL insulin syringe fitted witha 28 gauge needle. The mixture was allowed to gel for about 15 minbefore injecting into a circular silicone mold (diameter=about 4 mm,height=about 2.5 mm) within a 24 well plate using a syringe pump(SP220I; World Precision Instruments) at a flow rate of about 0.05 mLmin⁻¹. Cell viability was determined using LIVE/DEADviability/cytotoxicity kit (Invitrogen) at about 20 min post-injectionand about 3 d post-injection (n=5). Cells were fixed with about 4%paraformaldehyde, permeabilized with about 0.2% Triton X-100 solution inPBS, and stained with rhodamine phalloidin (1:300, Life Technologies)and 4′,6-diamidino-2-phenylindole (DAPI, about 1 μg mL⁻¹, LifeTechnologies). Images were collected using a Leica confocal microscopeby creating z-stacks of about 200 μm depth with about 2.4 μm intervalsbetween slices in the middle of the hydrogel and then compressing into amaximum projection image. Cell numbers were quantified using ImageJ ateach time point.

Statistical Analysis

Certain data in this example are presented as mean±standard deviation.Statistical comparisons were performed by one-way analysis of variance(ANOVA) with Tukey post hoc test. Values were considered to besignificantly different when the p value was <0.05.

TABLE 1 Reaction rates and equilibrium constants for hydrazone formationwith different concentrations of catalyst 1 calculated from modelreaction. Conc. of 1 (mM) k₁ (M⁻¹s⁻¹) k⁻¹ (s⁻¹) K_(eq) (M⁻¹) 0 5.44 4.29× 10⁻⁵ 1.27 × 10⁵ 25 1.27 × 10² 1.03 × 10⁻³ 1.24 × 10⁵ 50 2.50 × 10²2.03 × 10⁻³ 1.23 × 10⁵ 100 4.95 × 10² 4.01 × 10⁻³ 1.24 × 10⁵Conditions: about 137 mM NaCl, about 2.7 mM KCl, about 12 mM phosphate,pH of about 7.4 buffer, about 37° C.

As used herein, the singular terms “a,” “an,” and “the” may includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to an object may include multiple objects unlessthe context clearly dictates otherwise.

As used herein, the terms “substantially,” “substantial,” and “about”are used to describe and account for small variations. When used inconjunction with an event or circumstance, the terms can refer toinstances in which the event or circumstance occurs precisely as well asinstances in which the event or circumstance occurs to a closeapproximation. For example, when used in conjunction with a numericalvalue, the terms can encompass a range of variation of less than orequal to ±10% of that numerical value, such as less than or equal to±5%, less than or equal to ±4%, less than or equal to ±3%, less than orequal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%,less than or equal to ±0.1%, or less than or equal to ±0.05%.

As used herein, the term “alkyl” refers to a monovalent, aliphatichydrocarbon group. Examples include methyl, ethyl, propyl, isopropyl,butyl, and tertiary butyl.

As used herein, the term “alkylene” refers to a bivalent, aliphatichydrocarbon group. Examples include methylene, ethylene, propylene,isopropylene, butylene, and pentylene.

As used herein, the term “heteroalkylene” refers to an alkylene groupwhere one or more carbon atoms are replaced with an —O—, —S—, or —N(R)—group, where R is hydrogen, a C₁-C₆ alkyl group, or another substituentgroup.

As used herein, the term “C_(m)” when used with a group refers to mcarbon atoms in that group.

As used herein, the term “carboxyl” refers to —(C═O)—OH.

As used herein, the term “sulfonyl hydroxide” refers to —(SO₂)—OH.

Additionally, amounts, ratios, and other numerical values are sometimespresented herein in a range format. It is to be understood that suchrange format is used for convenience and brevity and should beunderstood flexibly to include numerical values explicitly specified aslimits of a range, but also to include all individual numerical valuesor sub-ranges encompassed within that range as if each numerical valueand sub-range is explicitly specified. For example, a range of about 1to about 200 should be understood to include the explicitly recitedlimits of about 1 and about 200, but also to include individual valuessuch as about 2, about 3, and about 4, and sub-ranges such as about 10to about 50, about 20 to about 100, and so forth. While this disclosurehas been described with reference to the specific embodiments thereof,it should be understood by those skilled in the art that various changesmay be made and equivalents may be substituted without departing fromthe true spirit and scope of this disclosure as defined by the appendedclaims. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, method, operationor operations, to the objective, spirit and scope of this disclosure.All such modifications are intended to be within the scope of the claimsappended hereto. In particular, while certain methods may have beendescribed with reference to particular operations performed in aparticular order, it will be understood that these operations may becombined, sub-divided, or re-ordered to form an equivalent methodwithout departing from the teachings of this disclosure. Accordingly,unless specifically indicated herein, the order and grouping of theoperations are not a limitation of this disclosure.

What is claimed is:
 1. A hydrogel composition comprising: a polymernetwork including a first water-soluble polymer and a secondwater-soluble polymer that are crosslinked through dynamic bonds; and acatalyst to modulate a rate of exchange of crosslinking of the polymernetwork, wherein the catalyst is an N-heterocyclic aromatic compoundsubstituted with an aminoalkyl group and an alkyl sulfonyl hydroxidegroup.
 2. The hydrogel composition of claim 1, wherein the firstwater-soluble polymer and the second water-soluble polymer arecrosslinked through hydrazone bonds.
 3. The hydrogel composition ofclaim 1, wherein the first water-soluble polymer is modified with ahydrazine or hydrazide functional group, and the second water-solublepolymer is modified with an aldehyde functional group.
 4. The hydrogelcomposition of claim 1, wherein the first water-soluble polymer and thesecond water-soluble polymer are selected from polysaccharides andproteins.
 5. The hydrogel composition of claim 1, wherein at least oneof the first water-soluble polymer or the second water-soluble polymeris hyaluronic acid.
 6. The hydrogel composition of claim 1, wherein thefirst water-soluble polymer is hyaluronic acid modified with a hydrazineor hydrazide functional group, and the second water-soluble polymer ishyaluronic acid modified with an aldehyde functional group.
 7. Thehydrogel composition of claim 1, wherein a concentration of the catalystin the hydrogel composition is in a range from 0.5 mM to 500 mM.
 8. Thehydrogel composition of claim 1, wherein a content of the polymernetwork in the hydrogel composition is in a range from 0.5 wt. % to 30wt. %.
 9. A method of therapeutic cell delivery comprising: providing afirst water-soluble polymer and a second water-soluble polymer;providing a catalyst; cross-linking the first water-soluble polymer andthe second water-soluble polymer through dynamic bonds to form ahydrogel comprising a polymer network; encapsulating cells in thepolymer network of the hydrogel; and injecting the hydrogel into asubject, wherein the catalyst facilitates gel shear-thinning andinjectability, but quickly diffuses out of the hydrogel after injection,thereby stabilizing the hydrogel and slowing hydrogel erosion afterinjection to provide a long-term, cell adhesive scaffold for cellculture.
 10. The method of claim 9, wherein injecting the hydrogelcomposition is performed using a syringe.
 11. The method of claim 9,wherein the catalyst is a heterocyclic aromatic compound.
 12. The methodof claim 9, wherein the catalyst is an N-heterocyclic aromatic compoundsubstituted with an amino-containing group and a sulfonylhydroxide-containing group.
 13. The method of claim 9, wherein thecatalyst is zwitterionic.
 14. A method of forming a hydrogelcomposition, comprising: providing a first water-soluble polymer, asecond water-soluble polymer, and a catalyst; and combining the firstwater-soluble polymer, the second water-soluble polymer, and thecatalyst in a liquid medium including water to form the hydrogelcomposition, wherein the first water-soluble polymer is modified with ahydrazine or hydrazide functional group, and the second water-solublepolymer is modified with an aldehyde functional group, wherein the firstwater-soluble polymer and the second water-soluble polymer arecrosslinked through hydrazone bonds to form a polymer network, and arate of exchange of crosslinking of the polymer network is modulated bythe catalyst, wherein the catalyst is an N-heterocyclic aromaticcompound substituted with an aminoalkyl group and an alkyl sulfonylhydroxide group.
 15. The method of claim 14, wherein the firstwater-soluble polymer and the second water-soluble polymer are selectedfrom polysaccharides and proteins.
 16. The method of claim 14, whereinat least one of the first water-soluble polymer or the secondwater-soluble polymer is hyaluronic acid.